foods

Article Effects of Radishes, Apples, and Pears on the Lactic Acid Bacteria and Nutritional and Functional Qualities of Flavored Soy Sauce

Ashutosh Bahuguna 1, Il Guk Jo 1, Jong Suk Lee 2 and Myunghee Kim 1,3,* 1 Department of Food Science and Technology, Yeungnam University, Gyeongsan 38541, Korea; [email protected] (A.B.); [email protected] (I.G.J.) 2 Division of Food & Nutrition and Cook, Taegu Science University, Daegu 41453, Korea; [email protected] 3 Research Institute of Cell Culture, Yeungnam University, Gyeongsan 38541, Korea * Correspondence: [email protected]; Tel.: +82-53-810-2958



Received: 1 September 2020; Accepted: 23 October 2020; Published: 28 October 2020


Abstract: Producers of soy sauce are constantly making efforts to improve the sensory quality and nutritional value of their products. In this study, radishes, apples, and pears were used to prepare a distinctly flavored soy sauce, and the lactic acid bacteria, volatile compound content, and nutritional and functional qualities of the product were compared with two commercial flavored soy sauce products. Comparable physiochemical properties, antioxidant activities (in vitro and cellular), and higher prevalence of lactic acid bacteria (7.74 0.55 log CFU mL 1) were observed in ± − the prepared flavored soy sauce than in commercial flavored soy sauce. The comprehensive enzyme activity profile of the isolated lactic acid bacteria, Tetragenococcus halophilus (NCBI GenBank Accession no. MN270899), revealed the absence of any harmful enzymes such as β-glucuronidase. Moreover, the cell-free extract of T. halophilus showed xanthine oxidase inhibitory activity (half maximal inhibitory 1 concentration (IC50) = 0.79 mg mL− ), suggesting that the product possessed functionality against xanthine oxidase-induced oxidative stress. Additionally, the prepared flavored soy sauce had higher 1 1 amounts of total free amino acids (48.68 mg mL− ) and organic acids (7.77 mg mL− ). These results suggest that radishes, apples, and pears at a defined ratio are suitable for the large scale production of a flavored soy sauce with improved nutritional and functional qualities.

Keywords: cellular antioxidant; fermentation; flavored soy sauce; free amino acids; GC-MS; lactic acid bacteria; Tetragenococcus halophilus

1. Introduction Soybeans (Glycine max), which are a rich source of nutrients, are consumed whole or in the form of fermented products, such as Korean gochujang (a red chilli paste), doenjang (a brined soybean paste), chungukjang (a seasoning paste), and kanjang (soy sauce) [1,2], Japanese miso (a seasoning paste), and Indonesian tempeh (a compressed soybean cake) [3]. In particular, dark-colored salty soy sauces are the most popular fermented products in East Asian countries and are gaining acceptability worldwide due to their unique tastes, aromas, and health benefits [4]. Soy sauce contains various bioactive components exhibiting numerous properties such as anti-carcinogenic, antimicrobial, antiplatelet, and immunomodulating activity [5]. The preparation of soy sauce differs between regions based on the selection of raw materials, microbial starter cultures, strength of the brine, and time and temperature of the fermentation process, which eventually forms soy sauce with distinct properties [6]. In Korea, soy sauce is prepared by either the traditional method or the industrial method. In the traditional method, boiled soybeans are used to produce meju, with the fermentation being carried out by naturally evolved microorganisms.

Foods 2020, 9, 1562; doi:10.3390/foods9111562 www.mdpi.com/journal/foods Foods 2020, 9, 1562 2 of 19

By contrast, the industrial method uses defatted soybeans and wheat flour to produce the meju, with the inoculation of Aspergillus oryzae and Bacillus species [7]. Further, in both processes, meju is soaked in the brine solution and fermented for several months to create the soy sauce [8]. Soy sauce prepared in the traditional way is generally preferred by consumers [7,9], due to the unique taste and aroma imparted by the activities of a wide variety of naturally occurring molds (Aspergillus flavus, A. fumigatus, A. oryzae, Mucor adundans, Rhizopus nigricans, R. oryzae, and R. stolonifer), yeasts (Candida edax, Saccharomyces cerevisiae, and S. kluyveri), and bacteria (Bacillus citreus, B. circulans, B. licheniformis, B. megaterium, B. mesentericus, and Lactobacillus species) [8]. The high usage of soy sauce urges companies to enhance the quality in terms of sensory and nutrition. Most of the recent studies have focused on the development of new microbial strains and the optimization of the fermentation process to improve the production and functionalities of soy sauces [10]. Along with these techniques, the inclusion of other ingredients is emerging as a new trend for developing novel soy sauces with better sensory and functional attributes. Apples, pears, and radishes are rich sources of dietary supplements and have unique tastes and aromas. They contain important phytochemicals, such as carotenoids, flavonoids, isoflavonoids, and phenolic acids, which have many beneficial effects on human health [11–13]. Apples, in particular, are extensively consumed worldwide and have been linked in epidemiological studies to a decreased risk of diabetes, asthma, and cardiovascular diseases [12]. In this study, several flavored soy sauce products were prepared at laboratory scale by adding mixtures of radish, apple, and pear (at various proportions) and examined for quality. The best combination was then selected for production of the flavored soy sauce at plant scale (hereinafter PFSS) in order to evaluate the feasibility of its mass-level production and to compare with the two most popular commercial Korean flavored soy sauce products (hereinafter CFSS-A and CFSS-B).

2. Materials and Methods

2.1. Chemicals and Reagents The soybeans (Korean origin, Daewon cultivar) were purchased from a local market in Cheongsong (36◦260 N, 129◦030 E), Gyeongsangbuk-do, Republic of Korea. The apples (Hongro cultivar), radishes (Palkwang cultivar), and pears (Singo pear cultivar) were purchased from local farms in Gyeongsan (35◦490 N, 128◦440 E), Gyeongsangbuk-do, Republic of Korea. Salt (Sinan, Jeollanam-do, Republic of Korea) was purchased from a supermarket in Gyeongsan. Standard sugars and sugar alcohols (sucrose, glucose, galactose, fructose, mannitol, and sorbitol) and organic acids (lactic acid, acetic acid, and succinic acids) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The high-performance liquid chromatography (HPLC)-grade solvents were purchased from Merck (Darmstadt, Germany). Unless otherwise stated, all other chemicals used were of the highest quality and used as supplied.

2.2. Preparation of Flavored Soy Sauces at Laboratory Scale The traditional Korean soy sauce was prepared with 32% soybeans meju in 18% salt solution and served as control. For the meju preparation, boiled soybeans were crushed and transferred into stainless steel square plates to make the soybean blocks and fermented at ambient temperature for 2 months by natural microflora. Source materials for the preparation of flavored soy sauce, such as pulverized radish, apple, and pear, were supplemented separately with the basic ingredients of traditional Korean soy sauce at 10, 30, and 30% proportion (w/w), respectively. Radish, apple, and pear were pulverized using the mechanical grinder. Radish, apple, and pear-supplemented soy sauces were further mixed in different proportion and naturally fermented for 6 months to produce 4 distinct types of flavored soy sauce (designated FSS-A through FSS-D; Table1) (Figure1 and Supplementary Materials S1.1). All the experiments were at least carried out in triplicate. Foods 2020, 9, x FOR PEER REVIEW 3 of 22

Foods4 distinct2020, 9, 1562types of flavored soy sauce (designated FSS-A through FSS-D; Table 1) (Figure 31 ofand 19 Supplementary Materials S1.1). All the experiments were at least carried out in triplicate.

TableTable 1. 1.List List of flavoredof flavored soy saucesoy sauce prepared prepared by mixing by radish,mixing apple, radish, and apple, pear in and diff erentpear proportions.in different proportions. Flavored Soy Sauce Was Prepared by Combining Various Traditional Korean Soy Flavored Soy Sauce FlavoredSauce Soy Sauce that WereWas Prepared Supplemented by Combining with PulverizedVarious Traditional Radish Korean (10%; w Soy/w ),Sauce that Flavored Soy Sauce (Sample Code) Were Supplemented with PulverizedApple (30%; Radishw/w (10%;), and w/w), Apple (30%; w/w), and (Sample Code) PearPear (30%; (30%;w w/w/w)) in in Di Differentfferent Proportions Proportions Control Traditional Traditional Korean Korean soy soy sauce sauce (without any any pulverized pulverized vegetable vegetable/fruit)/fruit) FSS-AFSS-A 1:1:1 1:1:1 (Radish (Radish added added soy soy sauce:pear sauce:pear addedadded soy soy sauce:apple sauce:apple added added soy soy sauce) sauce) FSS-BFSS-B 1:3:2 1:3:2 (Radish (Radish added added soy soy sauce:pear sauce:pear addedadded soy soy sauce:apple sauce:apple added added soy soy sauce) sauce) FSS-C 1:2:3 (Radish added soy sauce:pear added soy sauce:apple added soy sauce) FSS-C 1:2:3 (Radish added soy sauce:pear added soy sauce:apple added soy sauce) FSS-D 1:2:2 (Radish added soy sauce:pear added soy sauce:apple added soy sauce) FSS-D 1:2:2 (Radish added soy sauce:pear added soy sauce:apple added soy sauce) Traditional Korean soy sauce: 32% soybeans in 18% (w/v) salt solution. Traditional Korean soy sauce: 32% soybeans in 18% (w/v) salt solution.

FigureFigure 1. 1.Sequential Sequential stepssteps representingrepresenting thethe preparationpreparation ofof didifferentfferent flavoredflavoredsoy soy saucesauce (FSS-A,(FSS-A, FSS-B,FSS-B, FSS-C,FSS-C, and and FSS-D) FSS-D) at at laboratory laboratory scale scale and and preparation preparation of of flavored flavored soy soy sauce sauce at at plant plant scale scale (PFSS). (PFSS).

TheThe selectionselection ofof 10,10, 30,30, andand 30%30% proportionproportion ofof radish,radish, apple,apple, andand pearpear forfor thethe preparationpreparation ofof flavoredflavored soy soy sauce sauce was wasbased basedon on thethe preliminarypreliminary sensorysensory studystudy performedperformed withwith didifferentfferent proportionsproportions (0,(0, 10,10, 30,30, and 50%) of these these vegetable vegetable and and fruits fruits to to supplemen supplementt soy soy sauce. sauce. In the In thesensory sensory study, study, the thehighest highest preference preference was was accredited accredited for for 10% 10% radish radish and and 30% 30% for for each each apple apple and pear supplement soysoy saucesauce (Supplementary(Supplementary FigureFigure S1).S1). Therefore,Therefore, 10,10, 30,30, andand 30%30% proportionproportion ofof radish,radish, apple,apple, andand pearpear werewere selected selected for for the the final final preparation preparation of of flavored flavored soy soy sauce. sauce. 2.3. Determination of the pH, Total Soluble Solids, and Browning Potential of the Flavored Soy Sauces 2.3. Determination of the pH, Total Soluble Solids, and Browning Potential of the Flavored Soy Sauces The pH of the flavored soy sauce samples was evaluated monthly throughout the fermentation The pH of the flavored soy sauce samples was evaluated monthly throughout the fermentation process (0–6 months) using a pre-calibrated pH meter (Thermo Electron Corporation; Beverly, MA, USA). process (0–6 months) using a pre-calibrated pH meter (Thermo Electron Corporation; Beverly, MA, The content of total soluble solids was measured using a refractometer (Brinkmann Instruments Inc.; USA). The content of total soluble solids was measured using a refractometer (Brinkmann Rockaway, NJ, USA) after removal of the solid particles by centrifugation at 8000 g. The 0.2 µm Instruments Inc.; Rockaway, NJ, USA) after removal of the solid particles by centrifugation× at 8000× pore size membrane-filtered, 10-fold diluted flavored soy sauce samples (0–6 months) were subjected g. The 0.2 µm pore size membrane-filtered, 10-fold diluted flavored soy sauce samples (0–6 months) to a browning analysis by measuring the absorbance at 420 nm in a spectrophotometer (Tecan; Mannedorf, Switzerland). Foods 2020, 9, x FOR PEER REVIEW 4 of 22 were subjected to a browning analysis by measuring the absorbance at 420 nm in a spectrophotometer (Tecan; Mannedorf, Switzerland).

Foods 2020, 9, 1562 4 of 19 2.4. Sensory Evaluation Sensory evaluation with respect to flavor, salty taste, sweetness, and overall acceptability was 2.4. Sensory Evaluation examined by a panel of twenty trained people belonging to different age groups. The selection of panelistsSensory was evaluationmade based with on their respect sensitivity to flavor, towards salty taste, the 4 sweetness,tastes (sweet, and salty, overall bitter, acceptability and sour). The was panelistsexamined have by a experience panel of twenty and sufficient trained peopleexpertise belonging in conducting to different sensory age and groups. descriptive The selection analysis. of Beforepanelists starting was madethe analysis, based onpanelists their sensitivity discussed the towards taste properties the 4 tastes of (sweet, the food salty, samples. bitter, Plain and bread sour). andThe drinking panelists water have experience were served and to su eachfficient panelist expertise to rinse in conducting the mouth sensorywhile moving and descriptive from one analysis.sample toBefore another. starting Flavored the analysis, soy sauce panelists was discussedserved tothe each taste panelist properties in a of rand theomized food samples. manner. Plain Sensory bread evaluationand drinking for water each wereparameter served was to each judged panelist on a to7 rinsepoint the scale mouth [14]. while The sensory moving score from oneof numerical sample to valueanother. 1 represents Flavored soyextreme sauce dislike was served while to the each maximu panelistm numerical in a randomized value 7 manner. represents Sensory extreme evaluation like of thefor eachsample. parameter was judged on a 7 point scale [14]. The sensory score of numerical value 1 represents extreme dislike while the maximum numerical value 7 represents extreme like of the sample. 2.5. Preparation of the Flavored Soy Sauce at Plant Scale 2.5. Preparation of the Flavored Soy Sauce at Plant Scale The best combination of flavored soy sauce obtained from the laboratory scale preparations was selectedThe for best plant combination scale production of flavored in a soy100 sauceL porcel obtainedain jar from(Figure the 1) laboratory (Supplem scaleentary preparations Materials S1.1). was Theselected selection for plant of the scale best production flavored soy in sauce a 100 preparation L porcelain jarwas (Figure based1 on) (Supplementary the sensory qualities Materials (Figure S1.1). 2) andThe selectionphysiochemical of the best parameters. flavored soy Plant sauce scale preparation flavored wassoy basedsauce on fermentation the sensory qualitieswas carried (Figure out2 )for and 6 months.physiochemical Simultaneously, parameters. a traditional Plant scale Korean flavored soy soy sauce sauce without fermentation any addition was carried of radish, out for pear, 6 months. and appleSimultaneously, served as athe traditional control, Koreanwhile the soy two sauce most without popular any commercial addition of radish,Korean pear, flavored and applesoy sauces, served namedas the control, as CFSS-A while and the twoCFSS-B most (Supplementary popular commercial Materials Korean S1.2), flavored were soy used sauces, for namedthe comparative as CFSS-A assessmentand CFSS-B with (Supplementary respect to physicochemical Materials S1.2), properties were used (pH, for thetotal comparative soluble solids, assessment browning with potential), respect aminoto physicochemical nitrogen, sugars, properties organic (pH, acids, total free soluble amino solids, acids, browning volatile compounds, potential), amino antioxidants nitrogen, (in sugars, vitro andorganic cellular), acids, and free presence amino acids, of lactic volatile acid compounds, bacteria. antioxidants (in vitro and cellular), and presence of lactic acid bacteria.

FigureFigure 2. SensorySensory characteristics characteristics of of 4 4 different different flavored flavored soy soy sauces sauces after after 6 6 months months of of fermentation. fermentation. EachEach value value represents represents mean mean ± SDSD (n ( n= =20).20). Sensory Sensory characteristics characteristics we werere judged judged on ona 7 apoint 7 point scale scale (7: ± extremely(7: extremely like, like, 1: 1:extremely extremely dis dislike).like). Different Different lowercase lowercase letters letters on on a abar bar indicate indicate significantly significantly statisticalstatistical differences differences ( p < 0.05).0.05). 2.6. Determination of Amino Nitrogen, Free Sugars, Organic Acids, and Amino Acids 2.6. Determination of Amino Nitrogen, Free Sugars, Organic Acids, and Amino Acids Amino nitrogen was estimated using the formol titration method [15]. In brief, 0.5 mL of the Amino nitrogen was estimated using the formol titration method [15]. In brief, 0.5 mL of the flavored soy sauce sample was diluted 20 times with distilled water and shaken for 30 min. Then, flavored soy sauce sample was diluted 20 times with distilled water and shaken for 30 min. Then, 20 20 mL of 35% formalin was added, and the mixture was titrated against 0.1 N NaOH up to pH 8.5. mL of 35% formalin was added, and the mixture was titrated against 0.1 N NaOH up to pH 8.5. Quantification of the free sugars and organic acids was obtained by HPLC analysis [16]. For the Quantification of the free sugars and organic acids was obtained by HPLC analysis [16]. For the analysis of the free sugars and organic acids, the flavored soy sauce samples were diluted 4 times in analysis of the free sugars and organic acids, the flavored soy sauce samples were diluted 4 times in distilled water and filtered through a 0.45 µm syringe filter. Free sugars were quantified by injecting distilled water and filtered through a 0.45 µm syringe filter. Free sugars were quantified by injecting 20 µL of the filtered solution into an HPLC system coupled with a refractive index detector (Alliance, 20 µL of the filtered solution into an HPLC system coupled with a refractive index detector (Alliance, Water Co.; Milford, MA, USA). The column was eluted with a calcium-ethylenediaminetetraacetic acid Water Co.; Milford, MA, USA). The column was eluted with a calcium-ethylenediaminetetraacetic buffer solution (50 mg L 1) at a constant flow rate of 0.5 mL min 1. Identification and quantification acid buffer solution (50− mg L–1) at a constant flow rate of −0.5 mL min–1. Identification and of the different sugars were achieved by comparing the eluates against external sugar standards of known concentrations. The organic acids were quantified by injecting 20 µL of the flavored soy sauce Foods 2020, 9, 1562 5 of 19 sample into an HPLC system equipped with a refractive index detector (Shimadzu Co.; Kyoto, Japan), 1 with 5 mM H2SO4 used as the mobile phase at a constant flow rate of 0.5 mL min− . For estimation of the free amino acids, the flavored soy sauce samples were first diluted 50 times in 0.2 N NaOH and then filtered through a 0.45-µm syringe filter. Finally, 20 µL of the sample was injected into an amino acid autoanalyzer (Hitachi; Tokyo, Japan).

2.7. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis The different compounds present in the various flavored soy sauce samples were extracted using the solid-phase microextraction technique and then analyzed by GC-MS, according to the method described by Lee et al. [17] with modifications. In brief, 5 mL of the flavored soy sauce sample in a closed vial was heated for 10 min at 70 ◦C to evaporate the volatile compounds, which were subsequently adsorbed onto a carbowax/divinylbenzene polydimethylsiloxane solid phase microextraction fiber assembly (SPME). GC was conducted on an Agilent 7890B GC–5977 GC/MSD system equipped with an Agilent DB-WAX 92-122-7062E column (60 m 250 µm 0.25 µm; Agilent; Santa Clara, CA, USA). × × The temperature of both the injection port and column was maintained at 250 ◦C. Each sample was 1 injected at a spit ratio of 20:1, with a flow rate of 24 mL min− at 18.55 psi. The mass spectrometry readings were taken at 0.5-s scan intervals. The mass spectra of the compounds in the flavored soy sauce samples were compared with the spectra of known compounds stored in the National Institute of Standards and Technology MS library (2008 version).

2.8. Estimation of the Total Phenolics, Flavonoids, and In Vitro Antioxidative Activity The total phenolics in the flavored soy sauce samples were determined using the Folin–Ciocalteu method [18], whereas the total flavonoids were quantified according to the method described by Lamien-Meda et al. [19]. The in vitro antioxidative potential of the samples was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays [17].

2.9. Cellular Antioxidative Potential

2.9.1. Cell Culture and Cell Viability Assay Mouse fibroblast National Institute of Health (NIH) 3T3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (supplemented with 10% (v/v) fetal calf serum and a 1% penicillin-streptomycin 1 cocktail) at 37 ◦C in a 5% CO2 incubator. The effects of different concentrations (0–50 µL mL− ) of the flavored soy sauce samples on the viability of the NIH 3T3 cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

2.9.2. Determination of Cellular Reactive Oxygen Species (ROS) NIH 3T3 cells were grown in 24-well culture plates until 80% confluency and then treated with 1 1.5 µL mL− of the flavored soy sauce samples. After 24 h incubation, oxidative stress was induced in the cells by the administration of 0.25 mM H2O2 for 2 h. Finally, the cells were washed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and stained with 20 µM 20,70-dichlorofluorescein diacetate (DCFDA) for 30 min. After washing twice with 0.1 M PBS, the stained cells were visualized immediately under an epifluorescence microscope (Nikon; Kanagawa, Japan) for the production of reactive oxygen species. The fluorescence intensity was quantified using ImageJ software (National Institutes of Health; Bethesda, MA, USA).

2.10. Enumeration, Identification, and Enzyme Profiling of the Lactic Acid Bacteria The presence of lactic acid bacteria in the flavored soy sauce samples was determined by spreading different dilutions on de Man, Rogosa, and Sharpe agar medium (Difco; Sparks, MD, USA). The isolated strains of lactic acid bacteria were Gram stained to determine their size and shape. The bacterial cells Foods 2020, 9, 1562 6 of 19 were also processed for molecular characterization through 16S rRNA gene sequencing (Solgent Co., Ltd.; Daejeon, Korea). The 16S rRNA gene sequences were compared with those available on the GenBank database using the Basic Local Alignment Search (BLAST) tool, and a phylogenetic tree was constructed with the neighbor-joining method using MEGA6.0 software [20]. The enzymes produced by the isolated lactic acid bacteria were evaluated using the API ZYM Kit (bioMerieux; Marcy-l’Étoile, France) according to the manufacturer’s instructions.

2.11. Xanthine Oxidase Inhibitory Activity of the Lactic Acid Bacteria Tests on the xanthine oxidase inhibitory activity of the lactic acid bacteria were carried out using the cell-free culture supernatant and cell-free extract of the isolated bacteria. To prepare the cell-free culture supernatant, overnight-grown cultures of the lactic acid bacteria were centrifuged at 6000 g for × 10 min, the supernatant was collected, and its pH was adjusted to 7.4 with 0.1 N NaOH. The supernatant was freeze-dried and finally suspended in 0.1 M phosphate buffer (pH 7.8). To prepare the cell-free extract, the bacterial pellet was washed twice with 0.1 M phosphate buffer (pH 7.8) and sonicated for 10 min on ice at 3–10 s pulses. Finally, the cell lysate was centrifuged at 8000 g for 10 min and × processed for freeze drying. Inhibition of the enzyme activity was evaluated by mixing various concentrations of cell-free 1 1 culture supernatant or cell-free extract (0.25–2.5 mg mL− ) with 0.2 U mL− xanthine oxidase and 0.2 mM xanthine in 0.1 M phosphate buffer (pH 7.8). Finally, the absorbance was recorded at 290 nm. A control devoid of cell-free culture supernatant and cell-free extract was set up under the same experimental conditions. The percentage of xanthine oxidase inhibition was calculated with the following equation: inhibition (%) = (1 As/Ac) 100 (1) − × where As is the absorbance of the sample in the presence of cell-free culture supernatant or cell-free extract and Ac is the absorbance of the control.

2.12. Quantification of Biogenic Amines Biogenic amines in different flavored soy sauce samples were examined by the method adopted by Shukla et al., 2015 [16]. In brief, 1 mL of flavored soy sauce samples was mixed with 5 mL of 0.4 M perchloric acid and filtered through Whatman paper no. 1. The 1 mL of the filtrate was mixed with 200 µL of 2 M NaOH, 300 µL of a saturated solution of NaHCO3, and 2 mL of 0.04 M of dansyl chloride and incubated at 40 ◦C for 45 min. Subsequently, 100 µL of 25% NH4OH was added, and the volume of the mixture was adjusted to 5 mL using acetonitrile. Finally, the mixture was centrifuged at 2500 g for 5 min. The obtained supernatant was filtered through a 0.2 µm syringe filter and × processed for HPLC analysis (Dionex Ultimate 3000 UHPLC+ focused, Thermo Fisher Scientific; Waltham, MA, USA). The biogenic amines were separated over the C18 column (5 µm pore size, 4.6 mm internal diameter, 250 mm length) using 0.1 M ammonium acetate and acetonitrile mobile phase with gradient programming up to 35 min. The separated biogenic amine peaks were analyzed at 254 nm and quantified using the known standards.

2.13. Statistical Analysis All the experiments were carried out in triplicate, and the results are expressed as the mean ± standard deviation of three independent experiments. Statistical significance was calculated using one-way analysis of variance, followed by Duncan’s post-hoc test, using SPSS16 software (SPSS Inc.; Chicago, IL, USA). A value of p < 0.05 was considered as indicating statistical significance. Foods 2020, 9, 1562 7 of 19

3. Results and Discussion

3.1. Analysis of the Physicochemical Properties of the Flavored Soy Sauces Prepared at Laboratory Scale Four different types of flavored soy sauce were prepared using varying proportions of radishes, apples, and pears (Table1), and their physiochemical properties were analyzed. The pH is the most important factor in the soy sauce fermentation process for the commencement of several biochemical events. An acidic pH environment, ranging from 4.85 to 5.47, was recorded in all the flavored soy sauce samples after six months of fermentation (Table2). In all the samples, the pH value decreased progressively during the first two months of fermentation, likely as a result of the formation of various organic acids during the initial stage of fermentation [21]. However, the pH value increased after two months, suggesting proteolysis and the release of ammonia [22]. The pH changes during the fermentation process suggest the occurrence of various biochemical changes.

Table 2. Physiochemical analysis of different flavored soy sauce during the fermentation at laboratory scale.

Physicochemical Sample Fermentation Time (Months) Parameter Code 0 1 2 4 5 6 Control 5.37 0.00 NS 5.29 0.01 NS 5.39 0.00 NS 5.54 0.00 NS 5.37 0.00 NS 5.47 0.00 NS ± ± ± ± ± ± FSS-A 5.34 0.02 NS 5.10 0.00 NS 4.78 0.00 NS 4.94 0.0 NS 4.81 0.01 NS 4.94 0.01 NS ± ± ± ± ± ± pH FSS-B 5.34 0.01 NS 5.06 0.00 NS 4.65 0.00 NS 4.84 0.01 NS 4.71 0.01 NS 4.85 0.00 NS ± ± ± ± ± ± FSS-C 5.31 0.02 NS 5.09 0.01 NS 4.86 0.0 NS 5.20 0.01 NS 4.99 0.00 NS 5.12 0.01 NS ± ± ± ± ± ± FSS-D 5.35 0.03 NS 5.07 0.00 NS 4.95 0.01 NS 5.33 0.00 NS 5.14 0.01 NS 5.25 0.00 NS ± ± ± ± ± ± Control 38.07 0.06 a 38.33 0.57 a 38.77 0.20 a 39.40 0.00 a 39.83 0.06 a 40.60 0.00 a ± ± ± ± ± ± FSS-A 32.67 0.06 b 32.33 0.57 b 32.03 0.06 b 32.40 0.00 b 32.93 0.06 b 33.53 0.15 b ± b ± b ± b ± b ± b ± c Soluble solids ( Bx) FSS-B 32.53 0.06 31.00 0.00 30.73 0.00 30.63 0.06 31.43 0.12 31.83 0.06 ◦ ± ± ± ± ± ± FSS-C 32.20 0.10 b 31.33 0.58 b 30.23 0.06 b 30.23 0.06 b 30.90 0.10 b 31.60 0.00 c ± ± ± ± ± ± FSS-D 31.40 0.10 b 32.67 0.57 b 31.83 0.06 b 31.90 0.00 b 32.27 0.06 b 32.63 0.16 b,c ± ± ± ± ± ± Control 1.58 0.01 a 1.35 0.03 a 1.37 0.03 a 1.82 0.00 a 2.03 0.08 a 2.63 0.06 a ± ± ± ± ± ± FSS-A 1.38 0.01 b 1.04 0.03 b 1.12 0.02 b 1.38 0.00 b 2.00 0.03 a 2.62 0.05 a ± ± ± ± ± ± Browning potential FSS-B 1.35 0.01 b 1.05 0.04 b 0.99 0.02 b 1.17 0.00 c 1.64 0.03 b 2.14 0.05 c ± ± ± ± ± ± (A420 nm) FSS-C 1.34 0.01 b 0.99 0.03 b 1.05 0.03 b 1.40 0.10 b 1.80 0.03 a 2.24 0.04 b ± ± ± ± ± ± FSS-D 1.37 0.01 b 0.97 0.02 b 1.08 0.03 b 1.30 0.00 b 1.82 0.06 a 2.11 0.05 c ± ± ± ± ± ± Each value represents mean standard deviation of three independent experiments. Mean values followed by different superscripts (a–c) in± a column are significantly different. (p < 0.05). NS represent non-significant changes between the groups.

In addition to the pH, the quantity of soluble solids is an important physicochemical characteristic of fermented foods. Soluble solids comprise inorganic and organic materials, which vary greatly depending on the fermentation conditions [10]. The highest amount of soluble solids (40.60 ◦Bx) was detected in the traditional Korean soy sauce (control), whereas the lowest amount was found in FSS-C (31.60 Bx) after six months of fermentation (Table2). A negative correlation ( y = 0.517x + 45.7, ◦ − r2 = 0.89) between the total fruit or vegetable weight added and soluble solids content was observed for all the flavored soy sauce samples. The browning of soy products occurs through enzymatic and non-enzymatic processes, such as the Amadori rearrangement and the Maillard reaction [15,16]. All four flavored soy sauce products showed browning capacity, which increased with the progression of fermentation (Table2). These findings are supported by a similar study that showed the browning potential of edible insect-supplemented soy sauce [15].

3.2. Sensory Evaluation Sensory evaluation was carried out as a mandatory concern of foods toward its consumer acceptability, and the results are depicted in Figure2. There are many parameters to judge the sensory quality, which varies greatly with the types of food [16]. In all four combinations of flavored soy sauce, FSS-D achieved the highest sensory score for flavor (5.08 1.34), salty taste (5.16 1.09), and sweet taste ± ± (4.88 1.21), while the control traditional Korean soy accredited the least. Similar to this, a significantly ± Foods 2020, 9, 1562 8 of 19

better overall acceptability was observed for the FSS-D (5.81 0.72) (p < 0.05). Sensory results ± collectively acclaimed FSS-D as the best preparation. We speculate that the different sensory properties of the tested flavored soy sauces were due to the differences in their basic composition and microbial diversity in it that generated different end products such as organic acids, sugars, and amino acids which are the well-known sensory components responsible for sensory quality.

3.3. Comparative Study of Plant Scale Produced Flavored Soy Sauce with Commercial Flavored Soy Sauces At the laboratory scale, FSS-D proved to have good taste and sensory qualities (Figure2), while the physiochemical parameters were comparable with the traditional Korean soy sauce (control) and other three flavored soy sauce combinations (Table2). Hence, FSS-D was selected as the best combination for the mass-level production at the plant scale (for methodology refer to Supplementary Materials S1.1) and compared with the two most popular commercial Korean flavored soy sauces, named as CFSS-A and CFSS-B.

3.4. Analysis of the Physicochemical Properties An acidic pH in the range of 4.38 0.02 to 5.49 0.01 was observed in all the samples after six ± ± months of fermentation (Figure3A). The highest pH value was observed in the control soy sauce (5.49 0.01) and the lowest in PFSS (4.38 0.02). There was no significant difference in the pH among ± ± the PFSS (after six months of fermentation) and the two CFSS products. The soluble solids content varied significantly (p < 0.05) among the different soy sauces, with the highest amount measured in CFSS-B (61.66 0.58 Bx) and the lowest in CFSS-A (35.00 Bx) (Figure3A). The highest browning ± ◦ ◦ potential was observed in the control traditional Korean soy sauce (1.63 0.03), whereas the lowest ± was seen in PFSS (1.22 0.03) (Figure3B). Foods 2020, 9, x FOR PEER REVIEW ± 9 of 22

Figure 3. Comparison of the physicochemical properties of the final flavored soy sauce produced at plant scale (PFSS) with those of the control (traditional Korean soy sauce) andFigure commercial 3. Comparison flavored soy sauces of (CFSS-A thephysicochemical and CFSS-B). (A) pH and propertiessoluble solids. (B of) Browning the final potential flavored and amino soy nitrogen. sauce (C)produced Free sugars. (D at) Organic acids. plant scale (PFSS) with those of the control (traditional Korean soy sauce) and commercial flavored soy sauces (CFSS-A and CFSS-B). (A) pH and soluble solids. (B) Browning potential and amino nitrogen. (C) Free sugars. (D) Organic acids. Different lowercase letters on a bar indicate significantly differences (p < 0.05).

Foods 2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/foods The amino nitrogen content reflects the quality of soy sauce and is a common measure for assessing the progress of fermentation, particularly for protein-rich material. Moreover, it has a great impact on the flavor/aroma of the final product [23]. In China, the grading of soy sauce is based on the amino 1 nitrogen content, where products with a content higher than 0.7 g 100 mL− are considered to be Foods 2020, 9, 1562 9 of 19 the best (grade A) [10]. Traditional Korean soy sauce (control) showed the highest amount of amino nitrogen content (0.75 0.02%), followed by PFSS (0.71 0.01%), signifying intense proteolytic and ± ± deaminase activity leading to the formation of small peptides, amino acids, and ammonia. In contrast, commercial flavored soy sauce samples, CFSS-B (0.38 0.01%) and CFSS-A (0.17 0.01%), showed a ± ± significantly low amount of amino nitrogen (p < 0.05) (Figure3B).

3.5. Quantification of Total Sugars and Organic Acids Aside from having nutritional value, free sugars are key components that determine the flavor and taste of fermented soy products [16]. We assayed the soy sauces for the presence of sucrose, glucose, galactose, fructose, mannitol, and sorbitol and observed different amounts in the samples (Figure3C, Supplementary Figure S2). A markedly high amount of total free sugars was found in 1 1 CFSS-A (44.38 mg mL− ), followed by CFSS-B (5.19 mg mL− ), and the lowest amount was in PFSS 1 (0.05 mg mL− ). Among the different estimated sugars, glucose was present in all the samples with 1 1 abundance in CFSS-A (18.51 mg mL− ), followed by CFSS-B (1.80 mg mL− ), traditional Korean soy sauce 1 1 1 (control) (0.17 mg mL− ), and least in PFSS (0.05 mg mL− ) (Figure3C). Galactose ( 0.13 mg mL− ) and 1 sorbitol (0.02 mg mL− ) were present only in the control soy sauce and CFSS-B, respectively (Figure3C). Fructose, sucrose, and mannitol were detected only in CFSS-A and CFSS-B (Figure3C). A 31-fold higher 1 1 1 fructose (18.62 mg/mL− ), 2.4-fold higher sucrose (6.18 mg mL− ), and 5.4-fold higher (1.07 mg mL− ) mannitol was quantified in CFSS-A in contrast to CFSS-B. We speculate that the difference in the sugar content in all the soy sauce samples was due to the involvement of different microbial enzymes or the difference in the raw materials used for the preparation of the product. The diverse microbial population in the soy sauce acts on free amino acids and sugars, leading to the formation of various important organic acids that are not only responsible for the taste and aroma but also vital for many biochemical reactions and numerous health benefits [24,25]. Three different types of organic acids (namely, succinic acid, acetic acid, and lactic acid) were detected in the soy sauce samples (Figure3D, Supplementary Figure S3). The highest amount of organic acids was 1 1 1 observed in PFSS (7.77 mg mL− ), followed by the control (5.43 mg mL− ), CFSS-B (1.71 mg mL− ), 1 and CFSS-A (0.50 mg mL− ) (Figure3D). Results were allied well with the earlier performed free sugar results and exhibited a negative correlation between the amount of total free sugars and organic acids, suggesting the conversion of sugars to organic acids. Moreover, the role of the lactic acid bacterium Tetragenococcushalophilus in the production of various organic acids has been documented [21]. Furthermore, the higher amount of total organic acids in PFSS may be attributed to the presence of the added fruits and vegetables, which are considered as chief sources of organic acids or their precursor molecules [26]. Lactic acid was the most abundant of all the organic acids found, with the highest amount being 1 1 1 in PFSS (7.11 mg mL− ), followed by the control (4.95 mg mL− ), CFSS-B (0.89 mg mL− ), and CFSS-A 1 (0.21 mg mL− ) (Figure3D). The most probable reason for the di fferent lactic acid amounts among the different samples is the differences in the microbial populations (particularly lactic acid bacteria) that metabolize the simple sugars and convert them to organic acids and eventually effect the sensory and nutritional behavior of food. The presence of volatile acetic acid in soy products has been reported [27] and depends highly on the raw material used, pH, temperature, and microbial involvement [7]. Consistent with this, we also noticed the presence of acetic acid in all the soy sauce samples, with the highest amount being in 1 1 1 PFSS (0.47 mg mL− ), followed by CFSS-B (0.16 mg mL− ), CFSS-A (0.08 mg mL− ), and the control 1 1 (0.02 mg mL− ) (Figure3D). Interestingly, PFSS had the lowest amount of succinic acid (0.19 mg mL − ), 1 whereas CFSS-B had the highest (0.66 mg mL− ) (Figure3D).

3.6. Quantification of Free Amino Acids The amounts of total free amino acids in the different soy sauce samples are shown in Table3. Other than the nutrient value, amino acids are also accountable for taste and color of the foods [28]. Foods 2020, 9, 1562 10 of 19

1 The highest amount of total free amino acids was observed in the control (183.93 mg mL− ), followed by 1 1 PFSS (48.68 mg mL− ), and the lowest amount was observed in CFSS-A (24.18 mg mL− ). The difference in free amino acid profiles among the different soy sauces is likely due to the addition of different raw materials (substrates), which support the growth of different types of microbes with distinct proteases. The microbial action on the complex biomolecules, such as proteins, leads to the production of amino acids, which are one of the important constituents responsible for the sensory and nutritional quality of the foods [16]. We further characterized the free amino acids by dividing them into distinct groups on the basis of their taste (sweet, umami, bitter, and other) [16] and non-proteinogenic nature [29]. PFSS was found to contain a higher amount of sweet- and bitter-taste amino acids than CFSS-A and CFSS-B (Table3). Furthermore, amino acids responsible for non-taste were also examined, and the 1 1 highest amount was detected in control (65.97 mg mL− ), followed by CFSS-B (12.36 mg mL− ) and 1 1 PFSS (10.16 mg mL− ), while the least was detected in the CFSS-A (6.34 mg mL− ).

Table 3. Quantitative analysis of free amino acids in the plant scale produced flavored soy sauce (PFSS), traditional Korean soy sauce (control), and commercial flavored soy sauces (CFSS-A and CFSS-B).

Sample Amino Acid (mg mL 1) Traditional Korean − PFSS (a) CFSS-A (b) CFSS-B (c) Soy Sauce (Control) Threonine 2.28 0.31 1.04 1.17 Serine 3.40 ND (d) 1.49 2.33 Sweet taste Glycine 2.49 2.69 0.84 1.81 Alanine 6.80 8.38 1.58 2.06 Total 15.51 11.38 4.95 7.37 Aspartic acid 1.33 0.83 3.55 1.53 Umami taste Glutamic acid 14.53 0.87 3.09 15.17 Cystine 0.12 ND ND ND Total 15.98 1.70 6.64 16.70 Methionine 1.32 0.87 0.33 0.66 Isoleucine 4.45 3.46 1.52 1.26 Bitter taste Leucine 6.92 5.42 2.32 3.07 Lysine 67.58 5.83 1.25 1.17 Total 80.27 15.58 5.42 6.16 Tyrosine 0.85 0.19 0.28 1.02 Phenylalanine 4.53 2.97 1.60 2.21 Valine 4.68 4.55 1.65 1.4 Non-taste Histidine 0.36 0.02 0.28 0.54 Proline 54.88 2.41 1.40 5.34 Arginine 0.68 0.01 1.14 1.82 Total 65.97 10.16 6.34 12.36 Ornithine 2.61 1.51 0.13 0.25 β-Amino isobutyric acid 0.36 0.23 0.10 0.32 α-Amino adipic acid 0.88 0.32 0.06 0.13 Non-proteinogenic Citrulline 1.65 ND 0.23 ND amino acids γ-Amino-n-butyric acid 0.44 7.71 0.25 0.10 Ethanol amine 0.16 0.06 0.03 0.03 Hydroxylysine 0.09 0.04 0.02 0.03 Total 6.19 9.86 0.82 0.86 Grand total 83.93 48.68 24.18 43.46 (a) PFSS: Plant scale produced flavored soy sauce, (b) CFSS-A: commercial flavored soy sauce A, (c) CFSS-B: commercial flavored soy sauce B. (d) ND: not detected.

Amino acids belonging to non-proteinogenic types were also observed, whose essential biological role is well documented in the literature [29]. For example, γ-amino-n-butyric acid (GABA) is a well-known inhibitory neurotransmitter of the mammalian central nervous system [30] and a main 1 dietary supplement [31]. We observed the highest amount of GABA in PFFS (7.71 mg mL− ) and the Foods 2020, 9, 1562 11 of 19

1 least in CFSS-B (0.10 mg mL− ). We speculate the higher amount of GABA in PFSS was due to its microbial profile, particularly the lactic acid bacteria, whose important role as a producer of GABA has been well documented [32,33]. The total non-proteinogenic amino acids were measured highest in 1 1 PFSS (9.86 mg mL− ), followed by the control (6.19 mg mL− ), and the lowest amount being in CFSS-B 1 (0.86 mg mL− ) (Table3). The results collectively disclosed a good amount of non-proteinogenic and taste amino acids in PFSS, which could be effective in providing a specific taste to soy sauce along with the nutrient supplement.

3.7. GC-MS Profile of Flavored Soy Sauce The results of the GC-MS analysis reveal the presence of variety of flavor compounds, primarily belonging to alcohol, esters, acids, acetaldehyde, ketones, phenolics, furan, pyrazine, as well as sulfur containing and other constituents, in flavored soy sauce (Supplementary Figures S4–S7). The retention times and amounts (% area) of the identified compounds are presented in Supplementary Tables S1–S4. The highest number of volatile compounds were identified in the control (127), followed by CFSS-A (117), PFSS (111), and CFSS-B (103). These differences might be attributed to the variation in raw materials and also the involvement of microorganisms that utilize those materials to produce different metabolites [21]. Among the different microbes, T. halophilus is known to contribute to the production of aromatic organic acids, alcohols, esters, and aldehydes in soy sauce [21]. In the control sauce, benzenemethanol (an aromatic alcohol) was the predominant compound (with a peak area of 12.03%), which is well known for its pleasant aroma and a precursor for corresponding ester and acid, followed by 2-undecanol (8.31%) (a fatty alcohol and flavoring agent) and 2-undecanone (7.58%) (a ketone class) (Table S1) and is responsible for a strong odor. By contrast, benzeneethanol was predominant in PFSS, with a peak area of 17.17%. Aside from its high prevalence in ensuring a pleasant aroma, benzeneethanol also serves as a precursor molecule for the formation of 2-phenethyl esters, which are natural flavoring agents. Additionally, PFSS also contained tetramethylpyrazine (2.16%), whose supportive roles in blood circulation, heart ailments, and headache relief as well as antioxidative activity have been well documented in the literature [34]. A high presence of 3-methyl-1-butanol (5.12%) (commonly known as isoamyl alcohol and an important aroma and sensory compound [21]) was detected in PFSS compared with the control. Another flavor/aroma compound, benzaldehyde, was observed in all the samples, with CFSS-B having the highest amount (9.94%). Benzaldehyde is considered a key volatile compound in soy products and is responsible for woody and nasty odors [35]. All four soy sauce samples also contained 3-methylbutanal, whose role as a key aroma component has been well documented in the literature [36]. The highest amount of 3-methylbutanal was detected in the control (3.22%), followed by PFSS (2.91%). Interestingly, ethanol was detected as the major flavor/aroma compound of CFSS-A, with a peak area of 17.36% (Table S3), whereas the other samples had much lower quantities of this compound. The importance of furanones as the major flavor/aroma constituents of soy sauce has been shown by Harada et al. [37]. Consistent with this, we also noticed different types of furanones in the tested samples. A high amount of 2(3H)-furanone, dihydro-5-(2-octenyl) (Z) (6.65%) was found in PFSS (Table S2). In summary, the GC-MS analysis revealed the diverse profiles of flavor and non-flavor volatile metabolites in the soy sauces that could further influence the taste, aroma, and nutritional and functional attributes of the product. The variations in the metabolite profiles of the different soy sauces might be due to the selection of different raw materials, which have an impact on the growth of microorganisms and fermentation. In particular, a correlation between T. halophilus and increases in certain flavor/aroma compounds during fermentation has been documented [21]. Our results concur with the findings of many reports that show the presence of similar compounds in soy sauce samples [6,10,38].

3.8. Total Phenolics, Flavonoids, and In Vitro Antioxidative Activity Phenolics and flavonoids, which are secondary metabolites abundantly present in fruits and vegetables, have prominent health-promoting effects in humans. A high amount of phenolics were detected in CFSS-B Foods 2020, 9, x FOR PEER REVIEW 13 of 22

3.8. Total Phenolics, Flavonoids, and In Vitro Antioxidative Activity Phenolics and flavonoids, which are secondary metabolites abundantly present in fruits and

Foodsvegetables,2020, 9, 1562have prominent health-promoting effects in humans. A high amount of phenolics12 were of 19 detected in CFSS-B (2.42 ± 0.08 mg gallic acid equivalents (GAE) mL–1), followed by PFSS (2.20 ± 0.06 mg GAE mL–1), whereas the lowest amount was in the control (2.01 ± 0.01 mg GAE mL–1) (Figure 4A). (2.42 0.08 mg gallic acid equivalents (GAE) mL 1), followed by PFSS (2.20 0.06 mg GAE mL 1), These± results are in agreement with other published− data, suggesting that the amount± of phenolics− in whereas the lowest amount was in the control (2.01 0.01 mg GAE mL 1) (Figure4A). These results fermented soy products depends on the raw material± chosen [39]. −In addition to phenolics, a significantlyare in agreement high withamount other of publishedflavonoids data,were suggestingdetected in thatPFSS the (0.57 amount ± 0.01 ofmg phenolics quercetin in equivalents fermented mLsoy–1 products, p < 0.05) depends compared on with the rawthat materialin the control chosen and [39 CFSS]. In additionsamples to(Figure phenolics, 4B). a significantly high amount of flavonoids were detected in PFSS (0.57 0.01 mg quercetin equivalents mL 1, p < 0.05) ± − compared with that in the control and CFSS samples (Figure4B).

Figure 4. ComparisonComparison of of the the phenolics, phenolics, flavonoids, flavonoids, and and antioxidative antioxidative activity activity of the of thefinal final flavored flavored soy saucesoy sauce produced produced at plant at plant scale scale (PFSS) (PFSS) with with those those of ofcontrol control (traditional (traditional Korean Korean soy soy sauce) sauce) and commercial flavoredflavored soysoy saucessauces (CFSS-A (CFSS-A and and CFSS-B). CFSS-B). (A (A) Total) Total phenolics phenolics and and flavonoids, flavonoids, expressed expressed as 1 1 asgallic gallic acid acid equivalents equivalents (GAE) (GAE) mL mL− –1and and quercetin quercetin equivalents equivalents(QE) (QE) mLmL−–1 of soy soy sauce, sauce, respectively. respectively. (B) 2,2-Diphenyl-1-picrylhydrazyl2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ferricferric reducing antioxidantantioxidant powerpower assays.assays. The half

maximal inhibitory concentration (IC50)) value value corresponds corresponds to to the the concentration concentration achieving 50% DPPH radical-scavenging activity. Each value represents the mean standard deviation of three independent radical-scavenging activity. Each value represents the ±mean ± standard deviation of three independentexperiments. Valuesexperiments. with di ffValueserent lowercasewith different letters lowercase differ significantly letters differ from onesignificantly another (p from< 0.05). one another (p < 0.05). Numerous diseases, such as cancer and rheumatoid arthritis, are directly associated with ROS-inducedNumerous oxidative diseases, stress such as [40 canc]. Theer and clearance rheumatoid of ROS arthritis, is usually are directly carried outassociated by many with cellular ROS- inducedenzymatic oxidative and non-enzymatic stress [40]. The antioxidants. clearance of However, ROS is usually when carried a massive out amount by many of cellular ROS is enzymatic generated, andthe residentnon-enzymatic antioxidants antioxidants. may prove However, to be when insuffi a cientmassive for amount reducing of theROS ROS is generated, to a basal the level resident [40], antioxidantsand external may antioxidants prove to are be required.insufficient Hence, for reducing the intake the of ROS antioxidant-rich to a basal level foods [40], would and external provide antioxidantsan excellent wayare required. to nullify Hence, the challenges the intake posed of byantioxidant-rich ROS. In this study, foods thewould antioxidative provide an potential excellent of waythe soy to nullify sauce samplesthe challenges was examined posed by byROS. DPPH In this assay. study, PFSS the antioxidative was revealed potential as a potent of the scavenger soy sauce of 1 samplesDPPH radicals, was examined with the by lowest DPPH half assay. maximal PFSS inhibitorywas revealed concentration as a potent (IC scavenger50 = 16.70 of DPPH0.91 µ Lradicals, mL− ), ± 1 which was 1.17 times lower (p < 0.05) than the IC value of CFSS-A (19.50 0.80 µL–1 mL ) (Figure4B). with the lowest half maximal inhibitory concentration50 (IC50 = 16.70 ± 0.91± µL mL ), which− was 1.17 The better antioxidative activity of PFSS can be justified by the fact that it–1 contains radishes, apples, times lower (p < 0.05) than the IC50 value of CFSS-A (19.50 ± 0.80 µL mL ) (Figure 4B). The better antioxidativeand pears, which activity are richof PFSS sources can be of phytochemicals,justified by the fact whose that potentialit contains role radishes, as antioxidants apples, and has pears, been wellwhich documented are rich sources in the literatureof phytochemicals, [12,13]. Additionally, whose potential many role reports as suggestantioxidants that thehas antioxidant been well documentedactivity of organic in the acids literature [41,42 [12,13].] may be Additionally, a reason for themany better reports antioxidant suggest profiling that the ofantioxidant PFSS, due activity to high prevalenceof organic ofacids organic [41,42] acid may in it. be Meanwhile, a reason for the the FRAP better assay antioxidant results suggest profiling a strong of PFSS, electron-donating due to high capacity of CFSS-B (0.92 0.03), followed by CFSS-A (0.88 0.01) and PFSS (0.86 0.02) (Figure4B). prevalence of organic acid± in it. Meanwhile, the FRAP ±assay results suggest ±a strong electron- donating capacity of CFSS-B (0.92 ± 0.03), followed by CFSS-A (0.88 ± 0.01) and PFSS (0.86 ± 0.02) 3.9. Cellular Antioxidative Potential (Figure 4B). The effects of the flavored soy sauce samples on the viability of NIH 3T3 cells were determined by 1 3.9.the MTTCellular assay Antioxidative (Figure5A–D). Potential All the samples up to 2.5 µL mL− concentration had no significant adverse effect on the viability of the cells. Evaluation of the cellular antioxidative activity by DCFDA The effects of the flavored soy sauce samples on the viability of NIH 3T3 cells were determined staining revealed that PFSS, CFSS-A, and CFSS-B were capable of inhibiting ROS, which was evident by the MTT assay (Figure 5A–D). All the samples up to 2.5 µL mL–1 concentration had no significant from the reduced fluorescent intensities (85.3 5.1%, 87.9 4.1%, and 88.6 4.8%, respectively) ± ± ± relative to that of cells stimulated with H2O2 only (100%). By contrast, the control soy sauce did not show any significant cellular antioxidative activity (Figure6A,B). These results suggest that PFSS is a better antioxidative product than the traditional Korean soy sauce and CFSS products. The most Foods 2020, 9, x FOR PEER REVIEW 14 of 22

adverse effect on the viability of the cells. Evaluation of the cellular antioxidative activity by DCFDA staining revealed that PFSS, CFSS-A, and CFSS-B were capable of inhibiting ROS, which was evident from the reduced fluorescent intensities (85.3 ± 5.1%, 87.9 ± 4.1%, and 88.6 ± 4.8%, respectively) Foodsrelative2020, 9to, 1562 that of cells stimulated with H2O2 only (100%). By contrast, the control soy sauce did not13 of 19 show any significant cellular antioxidative activity (Figure 6A,B). These results suggest that PFSS is a better antioxidative product than the traditional Korean soy sauce and CFSS products. The most probableprobable reason reason forfor this is is the the high high amount amount of oforga organicnic acids acids in PFSS in PFSS and the and presence the presence of the fruits of the and fruits andvegetables, vegetables, which which are aremajor major sources sources of ofmany many impo importantrtant phytochemicals phytochemicals that that are areresponsible responsible for for impartingimparting numerous numerous beneficialbeneficial eeffectsffects by directly directly scavenging scavenging ROS ROS or or inducing inducing cellular cellular antioxidants. antioxidants. ThereThere are are a a limited limited number number ofof publishedpublished studies studies on on the the cellular cellular antioxidative antioxidative potential potential of soy of soysauce. sauce. However,However, in in one one such such studystudy performedperformed on a a neuronal neuronal cell cell line, line, the the antioxidative antioxidative activity activity of bamboo of bamboo salt-supplementedsalt-supplemented soy soy sauce sauce in in inhibiting inhibiting ROS ROS was wa demonstrateds demonstrated by DCFDAby DCFDA staining staining [43 ].[43]. In a In similar a studysimilar by study Jeong by and Jeong Om [and44], Om the [44], protective the protec roletive of di rolefferent of different soy sauces soy against sauces against H2O2-induced H2O2-induced oxidative stressoxidative was demonstratedstress was demonstrated in U373MG in humanU373MG astrocytes. human astrocytes.

FigureFigure 5. 5.E Effectsffects ofof flavoredflavored soysoy sauce samples on on the the vi viabilityability of of NIH NIH 3T3 3T3 fibroblasts, fibroblasts, evaluated evaluated by by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidebromide (MTT)(MTT) assay.assay. The The control control represents represents cells withoutcells without any soy any sauce soy treatment. sauce treatment. (A) Traditional (A) Traditional Korean soyKorean sauce. soy (B sauce.) Flavored (B) Flavored soy sauce soy produced sauce at plantproduced scale (PFSS).at plant (C ,Dscale) Commercial (PFSS). (C flavored,D) Commercial soy sauces, flavored CFSS-A soy and sauces, CFSS-B, CFSS-A respectively. and CFSS-B, Each value representsrespectively. the Each mean valuestandard represents deviation the mean of ± threestandard independent deviation of experiments. three independent Values experiments. with different ± lowercaseValues with letters different differ lowercase significantly letters from differ one significantly another (p < from0.05). one another (p < 0.05).

3.10. Enumeration, Identification, and Enzyme Profiling of Lactic Acid Bacteria The roles of lactic acid bacteria in the fermentation of soy products are well documented in the literature, where they have a direct impact on the functional properties of food [45]. A significantly higher population of lactic acid bacteria were observed in PFSS (7.74 0.55 log CFU mL 1) than in the ± − control soy sauce (5.39 0.08 log CFU mL 1). Surprisingly, no lactic acid bacteria were observed in ± − CFSS-A and CFSS-B (Figure7A). On the basis of the colony morphology, size, shape, and Gram staining, only a single type of lactic acid bacteria was observed in the PFSS and control samples. Using comparative phylogenetic analysis based on 16S rRNA gene sequencing, the lactic acid bacterium was further identified as the closest homolog of T. halophilus strain JCM 5888 (98% similarity) (Figure7B). PFSS was supplemented with vegetables and fruits, such as apples, which also contain lactic acid bacteria [46]. However, it is unlikely that these native lactic acid bacteria appeared in the PFSS as soy sauce contains a high salt concentration (15–20%), which supports the idea that only the growth of halophilic lactic acid bacteria evolved during the fermentation. This corroborated the findings of a previous study that suggested a high prevalence of T. halophilus in soy sauce and other high-salted fermented products [47] and its involvement in the production of different organic acids and flavor/aroma compounds. The sequence of the isolated T. halophilus strain has been submitted to NCBI GenBank under the accession number MN270899. The high prevalence of T. halophilus in PFSS is correlated with our findings of a high amount of organic acids and diverse aroma compounds in this soy sauce product. Foods 2020, 9, 1562 14 of 19

Foods 2020, 9, x FOR PEER REVIEW 15 of 22

Figure 6. EffectsFigure of 6. flavored Effects of flavored soy sauce soy sauce samples samples on on th thee reactive reactive oxygen oxygenspecies level species in NIH 3T3 level in NIH 3T3 fibroblasts. (A) 2′,7′-Dichlorofluorescein diacetate (DCFDA) staining: (i) cells without H2O2 and soy A i fibroblasts. ( )sauce 20,7 0treatment-Dichlorofluorescein (control), (ii) cells diacetatestimulated with (DCFDA) 0.25 mM H staining:2O2 only, (iii () )cells cells treated without with H2O2 and soy sauce treatmenttraditional (control), Korean (ii) soy cells sauce stimulated + H2O2, (iv) cells with treated 0.25 with mM flavored H2O soy2 only, sauce produced (iii) cells at plant treated scale with traditional (PFSS) + H2O2, (v) cells treated with commercial flavored soy sauce-A (CFSS-A) + H2O2, and (vi) cells Korean soy sauce + H2O2,(iv) cells treated with flavored soy sauce produced at plant scale (PFSS) + treated with commercial flavored soy sauce-B (CFSS-B) + H2O2. (B) The fluorescence intensity H2O2,(v) cellsquantified treated withby Image commercial J software. Each flavored value repr soyesents sauce-A the mean (CFSS-A) ± standard +deviationH2O2 ,of and three ( vi) cells treated independent experiments. Values with different lowercase letters differ significantly from one with commercial flavored soy sauce-B (CFSS-B) + H2O2.(B) The fluorescence intensity quantified another (p < 0.05). by Image J software. Each value represents the mean standard deviation of three independent ± experiments.3.10. Values Enumeration, with Identific differentation, lowercase and Enzyme Profiling letters of di Lacticffer significantlyAcid Bacteria from one another (p < 0.05). Foods 2020, 9, x FOR PEER REVIEW 16 of 22 The roles of lactic acid bacteria in the fermentation of soy products are well documented in the literature, where they have a direct impact on the functional properties of food [45]. A significantly higher population of lactic acid bacteria were observed in PFSS (7.74 ± 0.55 log CFU mL–1) than in the control soy sauce (5.39 ± 0.08 log CFU mL–1). Surprisingly, no lactic acid bacteria were observed in CFSS-A and CFSS-B (Figure 7A).

Figure 7. Enumeration,Figure 7.identification,Enumeration, and xanthine identification, oxidase inhibitory and potential xanthine of lactic oxidase acid bacteria. inhibitory (A) Lactic acid potential bacterial populations of lactic acidin different bacteria. soy sauce samples. (B) Phylogenetic tree of the isolated lactic acid bacteria. The tree was constructed by comparing the 16S rRNA gene sequence of isolated lactic acid bacteria from plant scale produced flavored(A) Lacticsoy sauce acid (PFSS) bacterial with those of populations the other strains inpresent diff inerent database. soy (C sauce) Xanthine samples. oxidase inhibitory Values potential with of di theff erentcell-free lowercaseextract of the isolated lactic acid bacteria.letters The half maximal differ significantlyinhibitory concentration from (IC one50) was another determined (p with< 0.05). a logarithmic (B) Phylogeneticequation (y = 25.05 treeln(x) + of5.69). the Each isolated value represents lactic the acid mean ± standard deviation of three independent experiments. * Isolated lactic acid bacteria from flavored soy sauce sample. bacteria. The tree was constructed by comparing the 16S rRNA gene sequence of isolated lactic acid bacteria from plant scale produced flavored soy sauce (PFSS) with those of the other strains present in database. (C) Xanthine oxidase inhibitory potential of the cell-free extract of the isolated lactic acid

bacteria. The half maximal inhibitory concentration (IC50) was determined with a logarithmic equation (y = 25.05 ln(x) + 5.69). Each value represents the mean standard deviation of three independent ± Foods 2020, 9, x; doi: FORexperiments. PEER REVIEW * Isolated lactic acid bacteria fromwww.mdpi.com/j flavored soyournal/foods sauce sample. Foods 2020, 9, 1562 15 of 19

It is imperative that any microorganism used as a food additive must not produce any enzyme that is harmful to humans. Therefore, the comprehensive enzyme activity profile of the isolated T. halophilus was determined. This lactic acid bacterium produced beneficial enzymes, such as alkaline phosphatase, esterase, esterase lipase, and acid phosphatase (Table4). Importantly, it showed no activity for α-chymotrypsin, β-glucuronidase, and N-acetyl- β-glucosaminidase, which are usually associated with intestinal ailments [48].

Table 4. Enzymatic activity of the isolated Tetragenococcus halophilus (lactic acid bacterial strain) evaluated by the API ZYM kit.

Enzyme Substrate Enzyme Activity (nmol) Alkaline phosphatase 2-naphthyl phosphate >40 Esterase 2-naphthyl butyrate 20 Esterase lipase 2-naphthyl caprylate 20 Lipase 2-naphthyl myristate 0 Leucine arylamidase L-leucyl-2-naphthylamide 0 Valine arylamidase L-valyl-2-naphthylamide 0 Cystine arylamidase L-cystyl-2-naphthylamide 0 Trypsin N-benzoyl-DL-arginine-2-naphthylamide 0 α-Chymotrypsin N-glutaryl-phenylanine-2-naphthylamide 0 Acid phosphatase 2-naphtyl phosphate >40 Naphthol-AS-BI-phosphohydrolase naphthol-AS-BI-phosphate 5 α-Galactosidase 6-Br-2-naphthyl-αD-galactopyranoside 0 β-Galactosidase 2-naphthyl-βD-galactopyranoside 0 β-Glucuronidase naphthol-AS-BI-βD-glucuronide 0 α-Glucosidase 2-naphthyl-αD-glucopyranoside 0 β-Glucosidase 6-Br-2-naphthyl-βD-glucopyranoside 0 N-Acetyl-β-glucosaminidase 1-naphthyl-N-acetyl-βD-glucosaminide 0 α-Mannosidase 6-Br-2-naphthyl-αD-mannopyranoside 0 α-Fucosidase 2-naphthyl-αL-fucopyranoside 0

3.11. Xanthine Oxidase Inhibition Xanthine oxidase is an enzyme that catalyzes the conversion of hypoxanthine to xanthine and further to uric acid and superoxide radicals. Thus, an excessive activity of xanthine oxidase is associated with many pathological conditions [49]. The cell-free extract of the isolated T. halophilus displayed 1 xanthine oxidase inhibitory activity, with an IC50 value of 0.79 mg mL− (Figure7C). However, no inhibition of this enzyme was shown by the cell-free culture supernatant of the isolated bacterium. Although the inhibitory potential of the Lactobacillus species against many enzymes has been well documented in the literature [48], to the best of our knowledge, this is the first report showing such potential of T. halophilus against xanthine oxidase.

3.12. Quantification of Biogenic Amines The biogenic amines of the soy sauce samples are shown in Table5. The highest amount of the total biogenic amines was detected in PFSS (980.85 4.15 mg L 1) followed by the control ± − (891.11 12.17 mg mL 1), while the least was detected in CFSS-B (15.79 0.81 mg mL 1). Individually, ± − ± − tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermidine were quantified in each sample (Table5). The level of histamine in all the samples was in the range of 9.32 0.54 to 468.65 3.43 mg L 1 with a noteworthily high amount in control (468.65 3.43 mg L 1) ± ± − ± − and PFSS samples (461.99 0.99 mg L 1). Similar to histamine, putrescine was also detected in ± − abundance in control and PFSS samples. The minimum level of histamine and putrescine was noticed in CFSS-B, which was approximately 2-fold lower compared to the amount detected in CFSS-A (Table5). A diversified biogenic amine profile of the different samples was mainly due to the difference in raw Foods 2020, 9, 1562 16 of 19

material and subsequently the difference in microbial populations, which are the major producer of biogenic amines in fermented food [50,51].

Table 5. Quantification of biogenic amines in traditional Korean soy sauce, plant scale produced 1 flavored soy sauce (PFSS), and commercial flavored soy sauces (CFSS-A and CFSS-B) (mg L− ).

Biogenic Amine Traditional Korean Soy Sauce (Control) PFSS CFSS-A CFSS-B Tryptamine 18.19 0.00 13.92 1.19 6.13 0.07 0.96 0.01 ± ± ± ± 2-Phenylethylamine 20.69 0.02 24.68 0.77 2.16 0.03 1.68 0.02 ± ± ± ± Putrescine 348.28 7.54 438.20 0.42 3.74 0.01 1.73 0.03 ± ± ± ± Cadaverine 11.07 0.46 13.97 0.53 1.11 0.02 0.57 0.01 ± ± ± ± Histamine 468.65 3.43 461.99 0.99 18.68 0.9 9.32 0.54 ± ± ± ± Tyramine 23.49 0.66 27.85 0.16 3.10 0.33 0.73 0.02 ± ± ± ± Spermidine 0.67 0.04 0.24 0.09 2.35 0.32 0.62 0.13 ± ± ± ± Spermine 0.06 0.02 0.00 0.00 0.56 0.06 0.18 0.05 ± ± ± ± Total 891.11 12.17 b 980.85 4.15 a 37.27 1.74 c 15.79 0.81 d ± ± ± ± Each value represents mean standard deviation of three independent experiments. Mean values followed by different superscripts in a column± are significantly different (p < 0.05).

4. Conclusions On the basis of the above findings, we concluded that radishes, apples, and pears are suitable materials for influencing the soy sauce fermentation process and leading to the production of a flavored soy sauce with improved qualities. Our novel plant scale produced flavored soy sauce displayed a high content of flavonoids and a marked increase in antioxidants and lactic acid bacteria compared with the tested commercial flavored soy sauce products, suggesting that plant scale produced flavored soy sauce is more functionally active. Moreover, plant scale produced flavored soy sauce displayed a better profile of free amino acids, organic acids, and diverse volatile components due to its raw materials and distinct microflora and could be utilized for commercial production. Although there are few reports on the production of flavored soy sauce, to the best of our knowledge, this is the first comprehensive study demonstrating flavored soy sauce preparation by the addition of radish, apple, and pear and its comparative quality analysis.

Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/11/1562/s1, Figure S1: Sensory evaluation of the soy sauce supplemented with radish, apple, and pear. Sensory evaluation was conducted by 15 participants and preference for the food was given on a scale of 1-5. Numeric value 1 represents the highest preference while numerical value 5 represents the least preference towards the food; Figure S2: Chromatogram of free sugars detected in various soy sauce samples. (A) Traditional Korean soy sauce (control). (B) Plant scale produced flavored soy sauce (PFSS). (C) Commercial flavored soy sauce-A (CFSS-A). (D) Commercial flavored soy sauce-B (CFSS-B); Figure S3: Chromatogram of organic acids detected in various soy sauce samples. (A) Traditional Korean soy sauce (control). (B) Plant scale produced flavored soy sauce (PFSS). (C) Commercial flavored soy sauce-A (CFSS-A). (D) Commercial flavored soy sauce-B (CFSS-B); Figures S4–S7: Gas chromatography-mass spectrometry chromatograms of traditional Korean soy sauce (control), Gas chromatography-mass spectrophotometry chromatogram of plant-scale produced flavored soy sauce (PFSS), Gas chromatography-mass spectrophotometry chromatogram of commercial flavored soy sauce (CFSS-A), and Gas chromatography-mass spectrophotometry chromatogram of commercial flavored soy sauce (CFSS-B); Tables S1–S4: Gas chromatography-mass spectrometry profiles of compounds detected in traditional Korean flavored soy sauce (control), Gas chromatography-mass spectrophotometry profile of compounds detected in the plant scale produced flavored soy sauce (PFSS), Gas chromatography-mass spectrophotometry profile of compounds detected in the commercial flavored soy sauce (CFSS-A), and Gas chromatography-mass spectrophotometry profile of compounds detected in the commercial flavored soy sauce (CFSS-B). Author Contributions: Conceptualization, J.S.L. and M.K.; methodology, A.B., I.G.J., J.S.L., and M.K.; data analysis, J.S.L. and M.K.; funding acquisition, M.K.; resources, M.K.; supervision, M.K.; writing—original draft, A.B.; writing—review and editing, M.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was financially supported by the Ministry SMEs and Startups (MSS), Korea, under the “Regional Specialized Industry Development Program” (project no. R0005837), supervised by the Korean Institute of Advancement of Technology (KIAT) in years 2016–2017 and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A03044512) in 2020. Foods 2020, 9, 1562 17 of 19

Acknowledgments: The authors thank the Core Research Center for Natural Products and Medical Materials (CRCNM) for the technical support provided for the GC-MS analysis. Conflicts of Interest: The authors declare no conflict of interest.

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